Pharmaceutical compositions containing hyaluronic acid and collagenase for the topical treatment of wounds, burns and ulcers

ABSTRACT

The present invention concerns new compositions containing hyaluronic acid or the derivatives thereof in association with the proteolytic enzyme collagenase (and relative pharmaceutical formulations) for the preparation of a dressing for topical treatment of various kinds of wounds, burns of varying depth, pressure sores, vascular ulcers and diabetic foot ulcers as well as for the treatment of hypertrophic and keloid scars.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of application Ser. No.14/481,615, filed on Sep. 9, 2014, which is a Divisional Application ofapplication Ser. No. 11/921,835, filed on Dec. 27, 2007 (now U.S. Pat.No. 8,900,576, issued Dec. 2, 2014), which was filed as PCTInternational Application No. PCT/EP2006/006616 on Jul. 5, 2006, whichclaims the benefit under 35 U.S.C. § 119(a) to Patent Application No.PD2005A000207, filed in Italy on Jul. 7, 2005, all of which are herebyexpressly incorporated by reference into the present application.

SUBJECT OF THE INVENTION

The present invention concerns new compositions containing hyaluronicacid or the derivatives thereof in association with the proteolyticenzyme collagenase (and relative pharmaceutical formulations) for thepreparation of a dressing for topical treatment of various kinds ofwounds, burns of varying depth, pressure sores, vascular ulcers anddiabetic foot ulcers as well as for the treatment of hypertrophic andkeloid scars.

BACKGROUND OF THE INVENTION

Enzymatic débridement of necrotic tissue involves the removal of suchtissue by means of the action of non-toxic enzymes that are able todegrade denaturated collagen, fibrin and elastin present in thedevitalised tissue, preserving the viable tissue.

This technique is preferable to mechanical/surgical débridement becauseit is more selective towards granulation tissue and is especiallysuitable in non-infected lesions such as skin ulcers of diverse etiologyand of varying depth.

The enzymatic formulations available on the market today contain theproteolytic enzyme collagenase (Noruxol® and Iruxol®) or are constitutedby an association of fibrinolysin and desoxyribonuclease (Elase®), allof which are effective (albeit with different results) in removingnecrotic tissue, purulent exudate and fibrin (Mekkes J. R., ArchDermatol Rese, 1998, 290:152-157).

Of great interest from the point of view of application are collagenasesproduced from bacteria of the Clostridium species, although collagenaseproduced from Vibrio alginolyticus (Achromobacter collagenase EC3.4.24.08; Borivoj k., Matrix Supplement, 1992, 1: 127-133; EP0115974B1) produced from a non-pathogenic strain with a specific activity thatis markedly superior to that of the enzyme produced from Clostridium,has been widely described and characterised (albeit not yet marketed).

Collagenase is a protein that is very unstable in aqueous solutions evenat low temperatures. Moreover, it can easily be denaturated by chelatingagents and various metal ions that can interact with the calcium ionthat is essential for the enzymatic activity of the molecule.

It is an enzyme that is extremely sensitive to chemical-physicalprocedures such as freezing, thawing, freeze-drying and drying,processes that are often necessary in the course of preparing finalpharmaceutical formulations.

Various different formulations have therefore been tested to find afinal composition that contains a stable, and therefore active,collagenase (EP0543521B1, patent application WO96/41870, WO93/00807,WO94/24273).

Albeit still at an experimental stage, the enzyme is used in injectableform to treat Dupuytren's contracture, a deforming condition of thefingers, while the use of collagenase is particularly important inreducing glaucoma, a disorder that causes excessive pressure in the eyewith possible damage to the optic nerve, linked with an abnormal depositof collagen within the duct that drains biological fluids from theanterior chamber of the eye.

The collagenase enzyme is mainly indicated in débridement of burns ofvarying depth, pressure sores, vascular ulcers and diabetic foot ulcers.Moreover, it is used to treat hypertrophic and keloid scars.

Proper wound healing requires a proper reepithelialisation phase, whichfollows the phase involving removing the eschar, possibly by surgicaland/or enzymatic débridement.

Collagen is the chief component of necrotic tissue and, consequently, itis fundamentally important to remove it in order to favourreepithelialisation of the wound. However, in the course of thisoperation it is necessary to protect the surrounding skin to avoidpainful irritative phenomena resulting from the use of collagenase, asthe enzyme is capable of degrading both denaturated and naturalcollagen, hydrolysing the peptide bonds of the amino acid chain.

During enzymatic débridement the wound area being treated does notdecrease in size, it may even increase. Therefore, when the eschar hasbeen removed, the newly-forming granulation tissue is exposed andconsequently open to dangerous bacterial infections that may jeopardisecomplete healing of the lesion.

The wound healing process is a complex phenomenon involving many typesof cellular and humoral factors, and many phases that can favour theformation of pathological scarring, such as hypertrophic and keloidscars.

Proper healing therefore requires the application of drugs to guide (andaccelerate) the wound healing process.

Scientific and patent literature contains ample descriptions and claimsnaming hyaluronic acid (HA) as the chief factor in the tissueregeneration processes (European patent application EP1196179).

Indeed, thanks to its special chemical-physical and biological features,hyaluronic acid participates in and modulates all the main sequentialphases of wound healing:

-   -   inflammation;    -   formation of granulation tissue;    -   reepithelialisation;    -   scar remodelling.

Owing to its chemical-physical properties, said polysaccharide controlstissue hydration, creating the correct microclimate necessary to fasthealing; moreover, its high viscosity protects the wound from possiblebacterial and/or viral infections.

Thanks to its biological properties, hyaluronic acid has provedeffective in protecting against free radicals, in controllinginflammatory processes and stimulating angiogenesis. Its role incontrolling the expression of cytokines and trophic factors has beendemonstrated, and in stabilising the granulation tissue by favouring andregulating the flow of fibroblasts and endothelial cells into the woundduring reepithelialisation.

Lastly, experimental data have demonstrated an involvement of hyaluronicacid in controlling keratinocyte proliferation and the deposit ofcollagen in the wound, thus reducing the formation of fibrotic tissueand, therefore, of pathological scarring (John Chen W. Y. et al., WoundRepair and Regeneration, 1999, 7:79-89).

HA is a hetero-polysaccharide composed of alternating residues ofD-glucuronic acid and N-acetyl-D-glucosamine. It is a straight-chainedpolymer with a molecular weight in the range of 50,000 to 13×10⁶ Da,according to the source from which it is obtained and the methods usedto prepare it.

It is present in nature in the pericellular gels, the fundamentalsubstance of the connective tissue in vertebrate organisms (of which itrepresents one of the chief components), in the synovial fluid ofjoints, in the vitreous humor and umbilical cord.

HA therefore plays an important role in the biological organism (besidesthose described above), as a mechanical support for the cells of manytissues, such as the skin, tendons, muscles and cartilage.

Said polysaccharide is known to be used as a vehicle for drugs ofvarious kinds, in simple associations or salified with hyaluronic acid,since its special properties of biocompatibility, biodegradability,non-immunogenicity, viscosity and hydratability make it particularlysuitable as a release system for drugs and molecules both at a topicaland systemic level (EP0197718B1, EP0445255B1).

Indeed, preclinical experiments with HA associated withanti-inflammatories (such as Diclofenac) for topical use havedemonstrated that HA significantly increases (compared to controls)absorption of the drug into the skin where, thanks to the specificaction of hyaluronic acid, it is compartmented to form a “reservoir”,minimising further absorption through the skin. The drug's action (andefficacy) is thus significantly increased (Brown M. B. et al., JEADV,2005, 19:309-318).

The Applicant, contrary to the above description of HA as a deliverysystem, has surprisingly discovered that an association between HAand/or the derivatives thereof with the enzyme collagenase determines aclear reduction in the enzyme's activity, thus enabling thedegradation/removal of the eschar with simultaneous formation ofgranulation tissue, thanks to the specific action of HA.

Moreover, said polysaccharide protects the healthy tissue surroundingthe lesion from the digestive action of collagenase, thus increasingpatient compliance with the product.

By slowing down the proteolytic activity of collagenase, hyaluronic acidmanifests properties that prove to be the absolute opposite of those ofthe delivery system described above, as known to an expert in thecurrent state of the art.

A further subject of the present invention is represented bypharmaceutical formulations of a lipophilic nature, containingcollagenase in association with HA, that enable the completestabilisation of the enzyme and, therefore, its maintenance in an activeform, at room temperature for prolonged periods of time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes and claims the association of HA and/orthe derivatives thereof with the enzyme collagenase in pharmaceuticalformulations of a lipophilic nature which, thanks to the presence ofspecial excipients/stabilisers, allow the proteolytic enzyme to remainstable at room temperature for prolonged periods of time.

Thanks to HA's special properties in tissue regeneration, enzymaticdébridement by the new enzyme/HA composition substantially modifies thephases of eschar removal and new tissue regeneration that normallyfollow treatment with collagenase alone, permitting the establishment ofnew phases that determine proper wound healing without the formation ofpathological scarring:

-   -   the degradation/removal of eschar occurs at the same time as the        formation of granulation tissue;    -   the formation of new connective tissue determines a reduction in        wound size after eschar removal (for the reasons given above)        and, hence, a significant decrease in the risk of bacterial        and/or viral infections;    -   the healthy surrounding tissue is protected from the digestive        action of collagenase thanks to HA;    -   eschar removal can be performed painlessly.

The claims of the new collagenase/HA formulations described above arepossible because HA has proved capable of modulating the proteolyticactivity of collagenase significantly slowing down its action duringenzymatic débridement of the necrotic tissue, as demonstrated hereafter.

The formulation of enzyme/HA is particularly indicated in thedébridement of burns of varying depth, pressure sores, vascular ulcersand diabetic foot ulcers, wounds of various nature and of differentsizes and depth and, moreover, in the treatment of hypertrophic andkeloid scars with final patient compliance with the product that isdecidedly greater than that with the enzyme alone.

Furthermore, said composition is also claimed in the treatment ofDupuytren's contracture and glaucoma.

The HA derivatives that can be used in the new formulations that are thesubject of the present invention are listed below:

-   -   1. HA salified with organic and/or inorganic bases with a        molecular weight of 50-730KDa (EP0138572 B1) or with a high        molecular weight of 750-1230 KDa, (EP 535200 B1);    -   2. Hyaff®: esters of HA with alcohols of the aliphatic,        araliphatic, cycloaliphatic, aromatic, cyclic or heterocyclic        series, with a percentage of esterification that may vary        according to the type and length of the alcohol used, between 1        and 100%, preferably between 50 and 75% (EP 216453 B1);    -   3. Hyadd™: amides of HA with amines of the aliphatic,        araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic        series, with a percentage of amidation of between 1 and 10%,        preferably 4% (EP 1095064 B1);    -   4. O-sulphatated derivatives of HA up to the 4th degree of        sulphatation (EP 0702699 B1);    -   5. ACP®: inner esters of HA with a percentage of inner        esterification between 0.5 and 10% and preferably 5% (EP 0341745        B1);    -   6. Deacetylated HA derivatives: derived from the deacetylation        of the N-acetyl-glucosamine fraction with a percentage of        deacetylation preferably between 0.1 and 30%, while all the        carboxy groups of HA can be salified with organic and/or        inorganic bases (EP1313772 B1);    -   7. Hyoxx™: percarboxylated derivatives of HA obtained from the        oxidation of the primary hydroxyl of the N-acetyl-glucosamine        fraction with a degree of percarboxylation of between 0.1 and        100% and preferably between 25 and 75%. All the carboxy groups        of HA can be salified with organic and/or inorganic bases        (patent application No. EP1339753).

The HA used in the present invention as such or in the preparation ofits derivatives may be derived from any source, for example it may beobtained by extraction from rooster combs (EP0138572 B1), byfermentation, or by technological means and its molecular weight mayvary between 400 and 3×10⁶ Da, in particular between 1×10⁵ Da and 1×10⁶Da, and more particularly still between 50,000 and 200,000 Da.

The concentration of the collagenase enzyme to be used in associationwith HA or the derivatives thereof may vary between 0.01 U and 100U/milligram of HA, preferably between 0.1 and 20 U and more preferablystill between 0.2 and 10 U/mg of HA.

The final concentration of HA or the derivatives thereof in the finalpharmaceutical composition may vary between 0.01 and 5% weight/weight ofthe final product, ranging preferably between 0.1 and 2% w/w, and morepreferably still between 0.2 and 0.4% w/w.

One unit of collagenase is defined as the quantity of enzyme thathydrolyses 1 nmole of PZ-Pro-Leu-Gly-Pro-D-Arg in one second at pH equalto 7.1 and at 37° C. (PZ=4-phenyl-azobenzyloxycarbonyl) (Wunsch E. etal, Physiol Chem, 1963, 333:149-151).

The collagenase preferably used or to be used in the compositions thatare the subject of the present invention is that produced from thenon-pathogenic micro-organism belonging to the Vibrio alginolyticus sub.Iophagus strain, NCIMB (National Collection of Industrial and MarineBacteria), strain number 11038, equivalent strain: LMG 3418.

It is a gram negative micro-organism that produces a collagenase with amolecular weight of from 90,000 to 110,000 Daltons, (IUBMB nomenclature:EC 3.4.24.3), stable in a pH range of 4.0 and 11.0 but with an optimalpH of between 6.0 and 8.0, stable at temperatures varying between 4 and40° C. with an optimal temperature of 37° C.

The enzyme is defined as a metalloendopeptidase because it breaks up thecollagen into peptide fragments, acting directly on its triple-chainprotein structure. The specific activity of the enzyme is inhibited bythe silver and copper salts and also by chelating agents such as EDTA,which bind the calcium ion necessary to collagenase activity.

Collagenolytic Activity of collagenase from Vibrio alginolyticus Versusthe Activity of the Same Enzyme Associated with Hyaluronic Acid

The aim of the study was to compare the activity of the enzyme as suchwith that of the new composition of collagenase/HA, to observe theinfluence of hyaluronic acid on the proteolytic activity of the sameenzyme.

Material:

-   -   collagen extracted from bovine skin at a concentration of 1        mg/ml (of buffer prepared as described hereafter) as a substrate        for enzymatic activity;    -   collagenase extracted from Vibrio alginolyticus tested at three        different concentrations of 0.33, 0.66 and 1.32 enzyme units;    -   collagenase extracted from Vibrio alginolyticus tested at the        same three concentrations of 0.33, 0.66 and 1.32 U but in        association with hyaluronic acid at the following ratios: 0.16        U/mg of HA, 0.33 U or 0.66 U/mg of HA (hence, 0.33, 0.66 and        1.32 U are associated with 2 mg of HA). In all three cases, the        concentration of the polysaccharide was kept constant.        Method of Enzymatic Digestion:

the collagen degradation process was performed at 37° C. for a timeperiod initially set at 90 minutes. Subsequently, the process wasrepeated at the same concentrations and at the same temperature, but thereaction was halted 4 and 12 hours after it was begun.

The digestion reaction occurred in Tris-HCL buffer, 0.05 M, containingCaCl₂ 0.01M at pH=7.4 to which the reaction substrate had been added,either collagenase itself or the enzyme associated with setconcentrations of HA as described above.

Results:

the enzymatic digestion mixture obtained (enzyme and relative collagendegradation products) was analysed on 7% polyacrylamide gel(electrophoresis: SDS-PAGE; Laemmli U. K., Nature, 1970, 680-685), tomap the various protein fragments separated according to their molecularweight, (MW) and stained with Comassie blue.

After the electrophoresis run, the collagen separates into subunits witha MW of about 100,000 Daltons with further peptide fragments with a MWof about 33,000 Daltons.

All the gels were loaded with molecular weight standards, withundigested collagen as positive control, with collagen degraded bycollagenase as such and relative enzyme separately loaded and, lastly,with collagen degraded by collagenase associated with HA and enzymealone.

FIG. 1 clearly shows that 0.33 U enzymes only partially degrade collagenin 90 minutes but, above all, that the presence of HA in thecollagenase/HA composition (at the same concentration of enzyme)modifies the collagenolytic action of the enzyme, reducing its activity.

FIGS. 2 and 3 further show the modulatory action exercised by hyaluronicacid on the enzyme: after 90 minutes 0.66 and 1.32 U of collagenasecompletely degrade the collagen while, especially at 0.66 U, HA slowsdown the collagenolytic action of the enzyme. The influence of saidpolysaccharide on the enzyme is even more evident with 1.32 U ofcollagenase.

FIGS. 4 and 5 show the situation after 4 and 12 hours: after 4 hours ofincubation of the enzyme (0.66 U) with collagen it is still possible toobserve the action of HA on collagenase because the undigested proteinfractions of collagen are visible, albeit faintly; after 12 hours (againwith 0.66 U of enzyme) the modulatory action of the polysaccharide is nolonger evident and the collagen is completely degraded.

For purely descriptive purposes, and without being limited by the same,we report hereafter some examples of how the new formulations that arethe subject of the present invention can be prepared:

Method of Producing Collagenase/HA Lipogel

Preparation of the Functional Components of the Lipogel:

An initial aqueous solution or a buffer solution at pH 7.1 (TrisHCL 25mM, CaCl₂ 10 mM) is prepared with 82.7 U/ml of collagenase; 45 ml ofthis solution is freeze-dried with the following excipients: Maltose (18g) as diluent/stabiliser, Carrageenan, possibly purified (0.54 g) asstabiliser and water 45 g. 18.6 g of freeze-dried product is obtained(dry product), composed as follows:

-   -   maltose 95% w/w    -   carrageenan 2.8% w/w    -   collagenase 0.9% w/w

The freeze-dried product is then micronised.

At the same time, HA obtained by fermentation and with a MW of 160 KDsis also micronised.

Dispersion of the Functional Components in the Lipophilic Base:

EXAMPLES 1 AND 2

The enzyme collagenase and HA prepared as described above are evenlydispersed in a lipophilic base constituted by the following excipients:

-   -   hydrogenated castor oil in powder form (with gelling action)    -   cetylstearylic alcohol (as agent of consistence)    -   stringy Vaseline (as lipophilic phase)    -   light Vaseline oil (as lipophilic phase)        Preparation of the Lipophilic Base:

dissolve and solubilise at about 88-90° C. the stringy Vaseline andcetylstearylic alcohol in the Vaseline oil.

Once a smooth, melted mass has been obtained, dissolve and solubilisethe hydrogenated castor oil powder in the newly formed lipophilic phase.

Once the mass is evenly melted, cool to 25-30° C.

Lipogel: Example 1A

% w/w Function (weight/weight) Main components Micronised HA 0.2Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenatedcastor Gelling 1.0 oil agent Cetylstearylic alcohol Agent of 5.0consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oilLipophilic Q b a 100 phase

Lipogel: Example 1B

% w/w Function (weight/weight) Main components Micronised HA 0.2Freeze-dried, 1.5 U/g micronised collagenase Excipients Hydrogenatedcastor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 5.0consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oilLipophilic Q b a 100 phase

Lipogel: Example 1C

% w/w Function (weight/weight) Main components Micronised HA 1Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenatedcastor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 5.0consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oilLipophilic Q b a 100 phase

Lipogel: Example 1D

% w/w Function (weight/weight) Main components Micronised HYAFF (50% 0.2esterified with benzyl alcohol) Freeze-dried, 1.8 U/g micronisedcollagenase Excipients Hydrogenated castor Gelling 1.0 oil agentCetylstearylic alcohol agent of 5.0 consistence Stringy VaselineLipophilic 36.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 2A

Function % w/w Main component Micronised HA 0.2 Freeze-dried, 1.8 U/gmicronised collagenase Excipients Hydrogenated castor Gelling 1.0 oilagent Cetylstearylic alcohol agent of 10.0 consistence Stringy VaselineLipophilic 34.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 2B

Function % w/w Main component Micronised HA 0.2 Freeze-dried, 2 U/gmicronised collagenase Excipients Hydrogenated castor Gelling 1.0 oilagent Cetylstearylic alcohol agent of 10.0 consistence Stringy VaselineLipophilic 34.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Dispersion of the Functional Components in the Lipophilic Base:

EXAMPLE 3

Incorporate and solubilise light Vaseline oil in Jojoba Glaze whileslowly stirring at 70-75° C. (lipophilic gelling system based on styrenecopolymers-propylene-butylene in Jojoba oil) until a smooth mass isobtained. Cool to room temperature and then add, while stirring,Micronised HA and micronised, lipophilic Collagenase as previouslydescribed, until the powders have been completely amalgamated in thelipogel.

Lipogel: Example 3A

Function % w/w Main component Micronised HA 0.2 Accessory componentFreeze-dried, 1.8 U/g micronised collagenase Excipients Jojoba Glaze LVLipophilic 80.0 phase viscous agent Light Vaseline oil Lipophilic Q.s a100 phase

Lipogel: Example 3B

Function % w/w Main component Micronised HA 0.2 Accessory componentFreeze-dried, 1.5 U/g micronised collagenase Excipients Jojoba Glaze LVLipophilic 80.0 phase viscous agent Light Vaseline oil Lipophilic Q.s a100 phase

Said formulations may contain pharmacologically and/or biologicallyactive substances such as, for example, antibiotics, antivirals, woundhealers, cytostatic/cytotoxic agents, anticancer drugs, hormones,steroid and non-steroid anti-inflammatory drugs, trophic factors andcytokines of various nature.

The invention being thus described, it is clear that these methods canbe modified in various ways. Such modifications are not to be consideredas divergences from the spirit and purpose of the invention and anymodification that would be evident to an expert in the field comeswithin the scope of the following claims.

EXPLANATIONS OF THE FIGURES

FIG. 1: Collagen digestion time of 90 minutes MWS=molecular weightstandards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenaseat a concentration of 0.33 U+collagen; Coll. 2=Collagenase at aconcentration of 0.33 U; Coll./HA 1=Collagenase at a concentration of0.33 U+collagen in the presence of HA; Coll./HA 2=Collagenase at aconcentration of 0.33 U in the presence of HA.

FIG. 2: Collagen digestion time of 90 minutes MWS=molecular weightstandards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenaseat a concentration of 0.66 U+collagen; Coll. 2=Collagenase at aconcentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at aconcentration of 0.66 U in the presence of HA.

FIG. 3: Collagen digestion time of 90 minutes MWS=molecular weightstandards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenaseat a concentration of 1.32 U+collagen; Coll. 2=Collagenase at aconcentration of 1.32 U; Coll./HA 1=Collagenase at a concentration of1.32 U+collagen in the presence of HA; Coll./HA 2=Collagenase at aconcentration of 1.32 U in the presence of HA.

FIG. 4: Collagen digestion time of 4 hours MWS=molecular weightstandards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenaseat a concentration of 0.66 U+collagen; Coll. 2=Collagenase at aconcentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at aconcentration of 0.66 U in the presence of HA

FIG. 5: Collagen digestion time of 12 hours MWS=molecular weightstandards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenaseat a concentration of 0.66 U+collagen; Coll. 2=Collagenase at aconcentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at aconcentration of 0.66 U in the presence of HA.

The invention claimed is:
 1. A method for treating Dupuytren'scontracture which comprises administering to a subject havingDupuytren's contracture an injectable form of a pharmaceuticalcomposition comprising hyaluronic acid salified with inorganic baseswith a molecular weight of 50-730 kDa and the proteolytic enzymecollagenase, wherein said collagenase is from the non-pathogenicmicro-organism belonging to the strain Vibrio alginolyticus sub.Iophagus; wherein the hyaluronic acid acts to reduce the rate of theproteolytic activity of the collagenase in the subject; wherein theconcentration of the proteolytic enzyme collagenase ranges between 0.1and 20 U/milligrams of said hyaluronic acid; and wherein theconcentration of said hyaluronic acid is between 0.1 and 2% w/w.
 2. Themethod according to claim 1, wherein said pharmaceutical compositionfurther comprises an additional pharmacologically and/or biologicallyactive sub stance.
 3. The method according to claim 1, wherein theconcentration of the enzyme collagenase is between 0.2 and 10U/milligrams of said hyaluronic acid.
 4. The method according to claim3, wherein the concentration of said hyaluronic acid is between 0.2 and0.4% w/w.
 5. The method according to claim 1, wherein said compositionfurther comprises maltose and/or carrageenan as a stabilizing agent. 6.A method for treating Dupuytren's contracture which consists ofadministering to a subject having Dupuytren's contracture an injectableform of a pharmaceutical composition comprising hyaluronic acid salifiedwith inorganic bases with a molecular weight of 50-730 KDa and theproteolytic enzyme collagenase, wherein said collagenase is from thenon-pathogenic micro-organism belonging to the strain Vibrioalginolyticus sub. Iophagus; wherein the hyaluronic acid acts to reducethe rate of the proteolytic activity of the collagenase in the subject;wherein the concentration of the proteolytic enzyme collagenase rangesbetween 0.1 and 20 U/milligrams of said hyaluronic acid; and wherein theconcentration of said hyaluronic acid is between 0.1 and 2% w/w.